We are studying the regulation of dihydrofolate reductase (DHFR) gene expression in cultured mouse 3T6 fibroblasts. We have shown previously that the expression of this gene is very low in resting 3T6 cells. When the resting cells are serum stimulated to re-enter the cell cycle, DHFR gene expression increases dramatically at the beginning of S phase. We have isolated a methotrexate-resistant 3T6 cell line that overproduces the enzyme and its mRNA by a factor of 300, and that regulates DHFR gene expression in a manner similar (if not identical) to normal 3T6 cells. We are using this overproducing cell line to study in more detail the molecular mechanism(s) responsible for increasing and decreasing DHRF gene expression. We will attempt to answer the following questions: 1. Do the content and rate of production of DHFR mRNA increase at the beginning of S phase? 2. Is the production of DHFR mRNA controlled at the level of transcription or by regulating the efficiency of processing of the initial transcription product into mature mRNA? 3. Does the stability of DHFR and/or its mRNA change during transitions between the resting and growing state? 4. Are increases or decreases in DHFR gene expression controlled by regulating the efficiency of translation of DHFR mRNA? 5. Is DHFR gene expression regulated in the same manner (and perhaps by the same control signals) as other S phase enzymes?